Title of Invention

Synthetic capasaicin analogs as TRPVl agonists. Field of the invention and related prior art

The transient receptor potential cation channel subfamily V member 1 (TrpVl), also known as the capsaicin receptor and the vanilloid receptor 1, is a protein that, in humans, is encoded by the TRPVl gene. It was the first isolated member of the transient receptor potential vanilloid receptor proteins that in turn are a sub-family of the transient receptor potential protein group. This protein is a member of the TRPV group of transient receptor potential family of ion channels.

The function of TRPVl is detection and regulation of body temperature. In addition, TRPVl provides a sensation of scalding heat and pain (nociception).

TRPVl is a nonselective cation channel that may be activated by a wide variety of exogenous and endogenous physical and chemical stimuli. The best-known activators of TRPVl are: temperature greater than 43 °C (109 °F); acidic conditions; capsaicin, the irritating compound in hot chili peppers; and allyl isothiocyanate, the pungent compound in mustard and wasabi. The activation of TRPVl leads to a painful, burning sensation. Its endogenous activators include: low pH (acidic conditions), the endocannabinoid anandamide, N-oleyl-dopamine, and N-arachidonoyl-dopamine. TRPVl receptors are found mainly in the nociceptive neurons of the peripheral nervous system, but they have also been described in many other tissues, including the central nervous system. TRPVl is involved in the transmission and modulation of pain (nociception), as well as the integration of diverse painful stimuli.

A review of TRPVl agonists are given by Kaneko, Y. et al British Journal of

Pharmacology (2014) 171 p 2474-2507.

The problem with many TRPVl agonists is their toxicity, and side effects. Different antagonist also have different selectivity and therapeutic effects.

Naturally occurring and isolated capsaicin have been shown to be a potent TRPVl agonist. However the substance is toxic. The available amounts of naturally occurring capsaicin is limited and expensive. Natural capsaicin extracts contains at least 3 isomers, which all have different chemical properties. The amounts of these isomers and type and amount of additional isolated compounds will also depend on the natural source and the isolation procedure. It is thus difficult to obtain capsaicin extracts with sufficiently uniform purity and composition for an intended use. Naturally occuring capsacin has proved difficult to synthezise in high quality and yields. aXichem is a global supplier of natural-analogue industrial compounds and their mission is to develop, patent and market natural- analogue substances. aXichem has been focusing on synthetic analogues of natural capsaicin. Phenylcapsaicin is not environmentally harmful and is thus a viable alternative to natural capsaicin within a wide range of uses. aXichem holds European (EP1670310) and US patents (US7446226) on synthetic capsaicin analogs, their synthesis and use as antifouling agent (WO2005025314). These synthetic low toxicity analogs of capsaicin can by synthezised with high purity with a reasonable cost and in high yield. Phenylcapsaicin has proved to be less toxic and far less irritating than natural capsaicin.

Summary of the invention

The invention relates to at least one chemical compound with the general formula (1)

i

wherein R is a substituent selected from the group of Ci-Cis alkyl, trifluoromethyl, C3-C12 cycloalkyl, phenyl, phenoxy, phenylthio, halogen; or tautomers or salts thereof, for use as TRPV1 agonist(s).

The inventions also relates to pharmaceutical compositions, formulations and compositions comprising compounds of formula (1) according to claim 1.

The invention also relates to compounds, formulations and pharmaceutical compositions for treatment of diseases as defined in the claims 7 and 8.

Detailed description of the invention The invention relates to at least one chemical compound with the general formula (1)

1

wherein R is a substituent selected from the group of Ci-Cis alkyl, trifluoromethyl, C3-C12 cycloalkyl, phenyl, phenoxy, phenylthio, halogen; or tautomers or salts thereof, for use as TRPVl agonist(s).

A preferred compound according to claim 1, is phenylcapsaicin.

The invention also relates to a pharmaceutical composition comprising a compound according to any of the claims 1 or 2 and optional carriers and adjuvants, for use as TRPVl agonist(s).

Further, the invention relates to a combination comprising a compound according to any of the claims 1 or 2 or a composition according to claim 3 with one or more additional antagonist(s) selected form the group consisting of resiniferatoxin, piperine, curcumin, capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin, homocapsaicin, nonivamide.

The invention also relates to a pharmaceutical composition according to claim 3 or a combination according to claim 4, wherein the concentration of a compound of formula (1) according to claim 1 is between 2 and 500 ppm, preferably between 2 and 50 ppm even more preferably between 2 and 20 ppm.

The invention also relates to a compound according to claim 1, a pharmaceutical composition according to claim 3 or a combination according to claim 5, for use as TRPVl agonists in animals, preferably humans.

A compound according to claim 1 a pharmaceutical composition according to claim 3 or a combination according to claim 4 for treatment or prophylactic use for pain, coughing, asthma, cancer, anxiety, cardiac hypertrophy, diabetes, obesity, metabolic disorders, irritable bowel syndrome (IBS), Ulcus colitis, Crohn's disease.

The invention also relates to a method for treatment of disorders using one or more compounds according to claim 1, a pharmaceutical composition according to claim 3 or a combination according to claim 4, wherein the disorders are pain, coughing, asthma, cancer, anxiety, cardiac hypertrophy, diabetes, obesity, metabolic disorders, irritable bowel syndrome (IBS), Ulcus colitis, Crohn's disease.

Formulations, compositions and administration

The synthetic capsaicin analogs can be used in combinations with other agonist as for instance given in the review by Kaneko, Y. et al British Journal of Pharmacology (2014) 171 p 2474-2507.

Pharmaceutical compositions for medical use as drugs can be prepared with excipients and adjuvants according to standard textbooks.

The compounds or combinations according to the invention can be administered in all normal ways except rectally, for instance by oral, dermal, injection or inhalation administration.

Figures

Figure 1. Dose response curves of phenylcapsaicin on TRPV1 and HEK 293.

Experimental/Results

Synthetic capsaicin as TPRV1 agonists

Effects of Phenylcapsaicin on TRPV1 Stably Expressed HEK293 cells using FLIPR:

The objective of this study was to test the effects Phenylcapsaicin on HEK293 cell line and TRPV1 (Transient receptor potential vanilloid 1) stably expressed cell line using FLIPR. In brief, TRPV1 cells were grown in a 384 well plate and loaded with Calcium 5, a fluorescent probe that reports changes in intracellular calcium. TRPV1 channel activity was assayed by measuring a baseline signal followed by application of TRPVl channel activator (agonist). Influx of calcium through TRPVl channels was detected as a rise in cytoplasmic calcium, which was reported as an increase in fluorescence.

HEK293 cells stably expressing the human TRPVl channel which under control of the T- REx promoter and induced with tetracycline and HEK293 parental cells were cultured in a humidified and air-controlled (5% C0 ₂ ) incubator at 37 °C. Culture media were as following:

pH is adjusted with 1M NaOH to pH7.4

Phenylcapsaicin were weighed and dissolved in DMSO as shown in below table:

Phenylcapsaicin (30mM stock) was diluted further into 6mM stock.

Cell preparation was initiated 1 day before the FLIPR experiment.

1. Passage cells when they were at approximately 80- 90% confluence.

2. Re-suspend pellet in lOmls of warm media, count cells and adjust to the appropriate concentration (5xl0 ⁵ /ml for TRPV1 and 4xl0 ⁵ /ml for HEK293) using warm media .

3. Induce channel expression: add tetracycline to the TRPV1 cell suspension to a final concentration of lug/ml in order to induce expression of the channel.

4. Plate out cells at 50ul/well in 384 well cell plates as shown below and incubate overnight in a humidified and air-controlled (5% C0 ₂ ) incubator at 37 °C.

Loading dye:

1. On the day of the assay, prepare loading buffer using Assay buffer according to the kit manual.

2. Remove media from cells by inversion the cell plate and centrifuge.

3. Add 30ul of loading dye per well to the cell plate.

4. Allow the cells to be incubated with dye for 1 hour in the dark at room temperature. Compound plate preparation:

The compound was diluted into DMSO and made stock solution of 6 mM. The stock solution were further diluted in DMSO in 3 fold series to make sub-stock solutions.

Immediately prior to running the assay, the stock and sub-stock solutions were diluted into assay buffer to achieve 3 x final concentration for testing in compound plate. There were total 11 concentrations tested. The top test concentrations on cells were 10 uM, the final DMSO concentration on cells was 0.13%. Meanwhile, 0.13% DMSO was used as negative control.

FLIPR reading:

1. After 1 hour loading, cell plate and compound plate were mounted to FLIPR for reading. Protocol details were as below:

Excitation wavelength (nm): 470-495 Emission Wavelength (nm): 515-575 Baseline read: samples 1-10 (Is a read)

Test compound addition after sample 10: 15μ1 compounds were added to 30μ1 cell plate giving 1:3 dilution

Post test-compound read: samples l l-180 (ls a read for samples 11-60, 2s a read for samples 61-180)

2. Data was automatically captured at the end of FLIPR experiment. The peak value of fluorescence signal (the maximum value) was used for data export after ' Subtract bias based on sample for further data analysis.

Data were analyzed using Excel 2007 and GraphPad Prism.

Results

The data of top concentration and DMSO control was used for z factor and S/N

calculation.

Dose response curves

Conclusions

1. Three data points were excluded and not used for data analysis which didn't affect the results.

2. Good z factors were achieved for Phenylcapsaicin on TRPV1 cells.

3. Phenylcapsaicin had potent agonist effect on TRPV1 cells. 4. The EC50 of Phenylcapsaicin on TRPV1 was 57.81 nM.

5. Phenylcapsaicin had no effect on HEK293 cells.

Toxicity studies

Comparative toxicity data between capsaicin and phenylcapsaicin:

Capsaicin and Phenylcapsaicin are rapidly absorbed from the gastro-intestinal tract and almost completely metabolized. The major route of excretion was biliary excretion and therefore, in the mass balance study, the majority of metabolites were detected in feces. Although the low recovery lowers the reliability of the study, derivation of reference values is still possible when considering a correction factor for systemic exposure. The metabolic pathways identified for Capsaicin and Phenylcapsaicin are identical, although the major metabolite is glucuronidated and oxidated for Phenylcapsaicin while the major metabolite of capsaicin is just glucuronidated. Tissue accumulation is unlikely to occur for both substances. In conclusion, the data support the read-across from capsaicin to close data gaps of phenylcapsaicin.

The 14C located on the carbonyl group could easily be lost from the fatty acid chain and transferred to 14C-acetyl coenzyme A upon hydrolysis of the amide linkage, which means the radioactivity generated from 14C could be transferred to numerous endogenous substances. The author of the study concluded that therefore un-assigned radioactive peaks on the radiochromatograms are most likely endogenous substances which acquired their radioactivity from the biochemical metabolism of 14Clabeled fatty acid chain. Such metabolism of 14C-labeled fatty acid chain could also explain the low recovery in the mass balance studies, as the biochemical product of fatty acid chain could either be numerous 14C -endogenous substances which are finally exhaled as 14C02. Low radioactivity in carcasses at the end of the study support thatl4C -phenyl-capsaicin and 14C-capsaicin was biotransformed to other 14C-labeled volatile organic compounds in multiple steps and these metabolites were gradually released as expired air via the lung. If needed correction of the internal reference values (AEL) using the total recovery (50%) seems to be valid as the majority of metabolites are excreted in bile. Only 2-3% parent were detected in bile. Thus systemic availablity of the a.s. is likely. A complete absorption of Capsaicin was also postulated by

Donnerer et al. 1990. Naunyn-Schmiedeberg's Arch. Pharmacol., 342, 357-361)

Acute Oral Tox - QECD 423: 2000 mg/Kg is applied by oral without produce mortality. Based on the test results, the test substance is unclassified according to the GHS. The acute oral toxicity LD50 value: LD50 > 5000 mg/Kg.

Acute Dermal Irritation/Corrosion - QECD 404:

The test item 0,5 mL was applied to the a gauze patch, then cover the gauze patch on a small area (approximately 6 cm ² ) of skin, which is held in place with non-irritating tape. The test item is non-irritationg to the rabbits skin within an observation period of up to 14 days starting after an exposure time of 4 hours.

Acute Eye Irritation/Corrosion - QECD 405:

0,1 ml of the test item was placed in the conjunctival sac of one eye of rabbit. The test substance is slightly irritant to the rabbit eye within an observation period of up to 7 days without washout after the test substance was placed in the conjunctival sac of one eye.

Acute Inhalation Tox - QECD 403, 2009:

Under the condtions of this study the single exposure acute inhalation LC50 of

phenylcapsaicin is not greater than 5.65 mg/L in male and female rats based on active substance.

Skin Sensitisation - QECD 406, 1992:

All animals survived throughout the test period without showing any clinical signs of toxicity. Under the conditions of the present study it can be stated that the test item

Phenylcapsaicin caused no reactions identified as sensitisation at the tested concentration. According to Commission Regulation (EU) No. 286/2011 as well as GHS (Globally Harmonized Classification System) the test item Phenylcapsaicin has no obligatory labelling requirement for skin sensitisation and is unclassified.

Reverse Mutation Assay - QECD 471, 1997:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Phenylcapsaicin did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Phenylcapsaicin is considered to be non-muytagenic in this bacterial reverse mutation assay.